PROJECT SUMMARY/ABSTRACT Substantial clinical and pathological variability has been reported in patients carrying an expanded repeat in chromosome 9 open reading frame 72 (C9ORF72) who are generally diagnosed with amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD). Emerging evidence suggests that C9ORF72 expression, RNA foci, and dipeptide-repeat proteins contribute to C9ORF72-related diseases; however, the majority of the clinico-pathological variability remains unexplained. We hypothesize that RNA sequencing (RNAseq) might reveal individual genes or groups of highly correlated genes (modules) that could account for this variability, which may help to identify much-needed druggable targets and/or biomarkers. Our preliminary studies performed in the frontal cortex, where we compared C9ORF72 expansion carriers to (disease) controls, demonstrated that the top differentially expressed gene was C9ORF72 itself. We also noticed that differentially expressed genes were enriched for the endocytosis pathway, which is consistent with the reported function of the C9ORF72 protein in vesicle trafficking. Furthermore, we detected a module enriched for vesicular pathways, and additionally, we discovered an association with survival after onset for several promising candidates involved in vesicular transport. To find new therapeutic targets and biomarkers, we are now proposing to increase the size of our study cohort and to examine another relevant region: the cerebellum. As such, we will perform RNAseq on a well-characterized cohort of C9ORF72 expansion carriers, patients without this expansion, and control subjects without neurological diseases. We will use multivariable linear regression models for gene- level analysis and weighted correlation network analysis for module-level analysis. Subsequently, we will evaluate genes and modules for associations with clinical and pathological features of the disease, including age at onset, survival after onset, C9ORF72 expression levels, RNA foci burden, and dipeptide-repeat protein levels (Aim 1). We will then assess whether similar findings can be obtained in other regions (e.g., frontal cortex and blood) as well as other diseases (e.g., Alzheimer?s disease and progressive supranuclear palsy), using existing RNAseq datasets. Additionally, we will validate our top differentially expressed genes and modules in our extensive cohort of clinical and pathological samples (Aim 2). The fact that we have one of the largest collections of C9ORF72 expansion carriers, combined with the wealth of clinico-pathological information we generated and the availability of existing RNAseq datasets from different regions and diseases, uniquely position us to perform this project, facilitating the discovery of novel therapeutic targets and potential biomarkers for C9ORF72-linked diseases.